Blue-white spot screening is a method for recombinant screening: Recombinants are screened based on the genetic characteristics of the vector, such as α-complement, antibiotic genes, and the like. Many of the vectors currently used carry a short segment of E. coli DNA with the regulatory sequence of the beta-galactosidase gene (lacZ) and the coding information for the first 146 amino acids. A multiple cloning site (MCS) was inserted into this coding region, which does not destroy the reading frame, but allows a few amino acids to be inserted into the amino terminus of β-galactosidase without affecting function. A host cell encoding a partial sequence of the C-terminal of β-galactosidase. Therefore, both the host and plasmid-encoded fragments have no enzymatic activity, but when they are present together, they form an enzymatically active protein. Thus, the lacZ gene is complementary to a host cell lacking a near-operator segment and a plasmid with a complete near-operator segment, termed a-complement. The LacZ+ bacteria produced by α-complementation produce blue colonies in the presence of the chromogenic substrate X-Gal under the action of the inducer IPTG, and thus are easily recognized. However, when the foreign DNA is inserted into the multiple cloning site of the plasmid, it almost inevitably results in an amino-terminal fragment having no α-complementing ability, so that the bacteria carrying the recombinant plasmid form white colonies. This type of recombinant screening, also known as blue-white screening. For example, after screening with blue and white spots, the calcified bacteria plate transformed by the ligation product was cultured in a 37 ° C incubator for 12-16 hr, and the bacteria having the recombinant plasmid formed white colonies.

The Lac+ bacteria produced by α-complementation are more easily recognized, and they are IPTG (isopropyl) in the presence of the chromogenic substrate X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside). Thiothio-β-D-galactoside induces the formation of blue colonies. When the exogenous fragment is inserted into the multiple cloning site of the vector, the reading frame is changed, the expressed protein is inactivated, and the resulting amino acid fragment loses α-complementation ability, so the transformant containing the recombinant plasmid is colored under the same conditions. Only white colonies can be formed on the induction medium. On the MacConkey medium, α-complementary Lac+ bacteria can decompose lactose in the McCain culture medium by β-galactosidase to produce lactic acid, which lowers the pH, thus producing red colonies, and when the exogenous fragment After the insertion, the α-complementing ability is lost, so that β-galactosidase is not produced, and the lactose in the medium cannot be decomposed, and the colony is white.

The molecular biology basis of blue-white spot screening is based on the positive and negative regulation mechanisms of the lactose operon:

1. Composition of lactose operon: E. coli lactose operon contains three structural genes of Z, Y and A, encoding galactosidase, peroxidase and galactosyl acetyltransferase, respectively, in addition to a manipulation sequence O, one Promoter P and a regulatory gene I. 2. Negative regulation of repressor protein: In the absence of lactose, the gene-encoded repressor protein binds to the manipulation sequence O, the lactose operon is in a repressed state, and cannot synthesize three enzymes that break down lactose; in the presence of lactose, lactose is induced The inducible repressor protein is allosteric and cannot bind to the manipulation sequence, and the lactose operon is induced to openly synthesize three enzymes that break down lactose. Therefore, this regulatory mechanism of the lactose operon is an inducible negative regulation. 3. Positive regulation of CAP: There is a CAP binding site upstream of the promoter. When E. coli changes from an environment with glucose as a carbon source to an environment with lactose as a carbon source, the concentration of cAMP increases and binds to CAP. CAP is allosteric, CAP binds to the CAP binding site near the lactose operon promoter sequence, activates RNA polymerase activity, promotes structural gene transcription, regulates protein binding to the operon and promotes transcription of structural genes, and positively controls the lactose operon. Regulate and accelerate the synthesis of three enzymes that break down lactose. 4. Coordination and regulation: The negative regulation of the repressor protein encoded by the I gene in the lactose operon and the positive regulation of CAP are coordinated and mutually restricted.

Can I use Matchmaker System Blue Spot screening with X-Gal instead of Xa-Gal?

No, X-Gal is different from Xa-Gal. X-Gal is the reaction substrate for E. coli β-galactosidase (lacZ), and Xa-Gal is the reaction substrate for yeast α-galactosidase (Mel 1p). Xa-Gal is used as a screening marker for blue-and-white screening in the Matchmaker Gold system . After protein interaction activates MEL1 gene expression, yeast cells can secrete and express Mel 1p.

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